铜绿假单胞菌PA2800在耐药性和致病性中的作用研究

发布时间：2018-01-15 12:32

本文关键词：铜绿假单胞菌PA2800在耐药性和致病性中的作用研究　出处：《西北大学》2010年硕士论文　论文类型：学位论文

【摘要】： 铜绿假单胞菌(Pseudomonas aeruginosa)是一种革兰氏阴性致病菌,在自然界中分布广泛并且致病率很高,是引起院内感染的三大病原菌之一。随着抗生素药物的广泛使用,铜绿假单胞菌多重耐药菌株和耐药率不断上升,多种抗生素失去其疗效,使得临床的抗感染治疗越来越困难。研究铜绿假单胞菌中的耐药基因及耐药机理对于控制铜绿假单胞菌感染具有重要的意义。在实验室的前期研究中,通过对铜绿假单胞菌转座突变体文库进行药物敏感性筛选,发现基因PA2800突变体对四环素和环丙沙星敏感性升高。本论文对PA2800基因进行了进一步深入研究。首先通过基因敲除的手段构建PA2800的基因敲除突变体,并与野生型铜绿假单胞菌比较,测定了其在固体和液体LB中抗生素抗性的变化,结果发现在固体和液体LB中PA2800突变体对四环素、环丙沙星和氯霉素敏感性都升高,证实PA2800基因与铜绿假单胞菌耐药性相关。研究抗生素对这一基因的调节发现,低于抑制浓度的四环素和红霉素对PA2800的表达具有激活作用,暗示这一基因有可能在抗生素存在下诱导铜绿假单胞菌产生抗性。同时,致病力比较研究发现PA2800和铜绿假单胞菌致病有关,用野生型PAO和PA2800突变体感染果蝇,突变体对果蝇的致死率更高。相关毒性因子在突变体和野生型中的表达测定表明,rhlR和znuA在PA2800突变体中的表达增强,这可能是导致突变体较野生型致死率高的因素。同时,我们还发现oprH在PA2800突变体中的表达增强。OprH是一个外膜孔蛋白,它的过量表达可以增强铜绿假单胞菌对喹诺酮类和氯霉素的摄取,所以oprH在突变体中表达得到增强也可能是导致突变体对坏丙沙星和氯霉素敏感性升高的原因之一。通过实验证实PA2800实突变体和野生型在运动性和生物被膜方面没有明显区别,说明PA2800基因与运动性和生物被膜生成没有直接关系。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a gram negative pathogen, widely distributed and very high morbidity in nature, which is one of three major pathogens of nosocomial infection. With the extensive use of antibiotics and multi drug resistant Pseudomonas aeruginosa strains and drug resistance rate is rising, many antibiotics lose its efficacy the anti infection treatment, more and more difficult. The clinical drug resistance gene and drug resistance mechanism of Pseudomonas aeruginosa is of great significance for the control of Pseudomonas aeruginosa infection.
In the preliminary research of the laboratory, drug sensitivity screening of Pseudomonas aeruginosa transposition mutant library showed that PA2800 gene mutants increased sensitivity to tetracycline and ciprofloxacin. The PA2800 gene was further studied in this paper.
First of all by knockout methods to construct PA2800 gene knockout mutant, and compared with the wild type Pseudomonas aeruginosa, the changes of antibiotics in solid and liquid LB resistance were determined. The results showed that in the solid and liquid LB in PA2800 mutant of tetracycline, ciprofloxacin and chloramphenicol sensitivity are increased, and confirmed that the PA2800 gene of Pseudomonas Pseudomonas resistance. Regulation of antibiotics for this gene discovery, subinhibitory concentrations of tetracycline and erythromycin on the expression of PA2800 with activation, suggesting that this gene may be induced by the presence of antibiotics in Pseudomonas aeruginosa resistance. At the same time, a comparative study of pathogenicity found PA2800 and Pseudomonas aeruginosa with pathogenic, wild type PAO and PA2800 mutant Drosophila mutants of Drosophila melanogaster infection, a higher mortality rate. The virulence factor in the mutant and the wild type in table As indicated that the expression of rhlR and znuA in PA2800 mutant, which may lead to mutant compared to the wild type lethal factors of high rate. At the same time, we also found that the expression of oprH in mutant PA2800 enhanced.OprH is an outer membrane protein, its overexpression can enhance the uptake of Pseudomonas aeruginosa to quinolones and chloramphenicol, so oprH expression is enhanced in the mutant may be one of the reason of bad ciprofloxacin increased mutant and chloramphenicol sensitivity. Experiments prove that PA2800 mutant and wild type were not significantly different between film movement and biological, PA2800 gene and motility and biofilm formation is not directly related.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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